(1) Field of the Invention
This invention relates to a cation exchanger, a process for producing the cation exchanger, and a packing for chromatography comprised of the cation exchanger.
(2) Description of the Related Art
Fine particles having a cation exchanging group are widely used in the field of liquid chromatography for analyzing or separating or isolating, for example, amino acids, peptide, protein, nucleic acids and saccharides.
Cation exchangers are usually made by introducing a cation exchanging group in fine particles by allowing the cation exchanging group to directly react with a functional group present on the surface of the fine particles. Specific examples of the method of introducing a cation exchanging group in fine particles, there can be mentioned a method of introducing a carboxylic acid by allowing, for example, chloroacetic acid to react with a hydroxyl group present on the surface of fine particles; and a method of introducing an allyl group or an epoxy group to a hydroxyl group present on a fine particle surface and then sulfonating the introduced group.
The cation exchanging group is introduced onto the surface of fine particles. When the fine particles used are porous, the cation exchanging group is introduced onto the inner wall of pores as well as the particle surfaces. Therefore, the amount of a sample to be analyzed or separated which is capable of being retained on the fine particle surfaces is determined principally on the surface area of the fine particles or on both of the surface area thereof and the pore diameter. The cation exchanger adsorbs or retains a sample to be analyzed or separated in a single layer form on fine particle surface. Therefore, when the cation exchanger is used for liquid chromatography, it rapidly absorbs or desorbs the sample and gives a sharp separation pattern.
In recent years, in order to enhance the rate of operation for separation of protein or other sample materials to be adsorbed, a cation exchanger having an improved capacity for adsorption of protein or the other materials is being developed. Attempts are being made for enhancing the adsorption capacity of fine particles. For example, a process for producing a cation exchanger capable of adsorbing a sample to form multi-layers of the sample on the fine particle surface by introducing a polymer chain having an ion exchanging group onto the fine particle surface has been proposed in U.S. Pat. No. 5,453,186. As the method of introducing the polymer chain having an ion exchanging group onto a fine particle surface, many proposals have been made, for example, in U.S. Pat. Nos. 3,723,306, ibid. 4,137,137, ibid. 4,298,698, ibid. 4,605,685, and ibid. 4,728,678. The proposed methods include (i) a method of graft polymerization, namely, irradiating a fine particle surface with radiation to form radical initiating sites, or treating a fine particle surface with a cerium salt or a manganese salt to form hydroxyl groups as radical initiating sites, and then, allowing a polymer chain to grow on each radical initiating site; (ii) a method of introducing an unsaturated group onto a fine particle surface and then treating the unsaturated group-introduced fine particle in a monomer solution whereby a polymer chain derived from the monomer is allowed to grow on the unsaturated group; and (iii) a method of introducing a functional group to a polymer terminal and then the polymer is allowed to react with a functional group present on a fine particle surface.
The cation exchangers produced by the above-proposed processes exhibit enhanced capacity for adsorbing an objective sample and, when the cation exchanger is used for separation of an objective sample, the operation can be carried out at a high rate. However, these cation exchangers have straight-chain polymers introduced onto the fine particle surfaces thereof, and therefore, when it is used as being packed in a column for liquid chromatography, a high operation pressure is required for feeding a liquid. Thus, pressure-resistant equipment and pipes must be used for liquid chromatography means spanning from a liquid-feed pump to a detector and for paths of flow connecting these means each other. Consequently problems arise in that the equipment cost increases, a sealing material is readily deteriorated and parts must be exchanged with a shortened interval of time, and further, liquid leakage tends to occur. Occasionally the cation exchangers are broken by the high pressure.
The liquid flow characteristics are closely related with the particle diameter of cation exchangers. If the particle diameter of cation exchangers is enlarged, the liquid passage characteristics can be improved, but, the separation capacity of cation exchangers as used for liquid chromatography is lowered. To sum up, the cation exchangers produced by the above-proposed processes cannot be advantageously employed for liquid chromatography for which a high adsorption capacity and a high separation capacity are required.
Another proposal has been made to improve the liquid passage characteristics by crosslinking polymer chains introduced onto the fine particle surface (for example, U.S. Pat. Nos. 4,100,149 and ibid. 4,376,047). However, it has not been reported in these patent publications whether the produced cation exchangers exhibit or do not exhibit enhanced capacity for adsorption of an objective sample to be analyzed or separated or isolated. It is presumed that a coating film layer (ion exchanging layer) comprised of the crosslinked polymer chains and formed on the fine particle surface is shrunk with the result of reduction of the adsorption capacity of an objective sample.
In view of the foregoing, an object of the present invention is to provide a cation exchanger comprised of a fine particle, which has an enhanced capacity for adsorption of a sample to be analyzed, separated or isolated, and exhibits improved liquid passage characteristics.
Another object of the present invention is to provide a process for producing a cation exchanger comprised of a fine particle, which exhibits an enhanced capacity for adsorption of a sample to be analyzed, separated or isolated and exhibits improved liquid passage characteristics.
Other objects of the present invention are to provide a packing for chromatography comprised of a cation exchanger exhibiting an enhanced capacity for adsorption of a sample to be analyzed, separated or isolated, and to provide a column for chromatography packed with the packing comprised of the cation exchanger.
In one aspect of the present invention, there is provided a cation exchanger comprised of a fine particle having bound to the surface thereof a polymer having a cation exchanging property, characterized in that said polymer having a cation exchanging property is at least one polymer selected from (i) polymers prepared by polymerization of at least one kind of unsaturated carboxylic acid and partially crosslinked with a polymer having a functional group capable of reacting with a carboxylic acid, and (ii) acrylic acid-maleic acid copolymers.
The polymers (i) are preferably copolymers prepared by copolymerization of at least one kind of unsaturated carboxylic acid selected from acrylic acid, methacrylic acid, maleic acid and itaconic acid with at least one kind of monomer selected from vinylsulfonic acid, 3-sulfopropyl acrylate, 3-sulfopropyl methacrylate, 3-sulfo-2-hydroxypropyl acrylate and 3-sulfo-2-hydroxypropyl methacrylate.
The polymer having a functional group capable of reacting with a carboxylic acid for partially crosslinking the polymer (i) preferably has at least one kind of functional group selected from a hydroxyl group, a glycidyl group and an amino group as the functional group capable of reacting with a carboxylic acid. The polymer having a hydroxyl group as the functional group capable of reacting with a carboxylic acid is preferably selected from polyvinyl alcohol and polysaccharides, and the polymer having an amino group as the functional group capable of reacting with a carboxylic acid is preferably polyethyleneimine.
The acrylic acid-maleic acid copolymers (ii) preferably have a weight average molecular weight of at least 10,000 and are bound to the fine particles through a single bond.
In another aspect of the present invention, there is provided a process for producing the above-mentioned cation exchanger, which comprises the steps oft dispersing fine particles in (i) a solution containing a combination of at least one polymer selected from polymers prepared by polymerization of at least one kind of unsaturated carboxylic acid, with a polymer having a functional group capable of reacting with a carboxylic acid, or (ii) a solution containing at least one acrylic acid-maleic acid copolymer, to prepare a dispersion of fine particles; removing a liquid medium from the dispersion of fine particles to separate a solid content; and then, heating the separated solid content in a dried state to obtain a fine particle having bound to the surface thereof a polymer having a cation exchanging property.
The fine particles preferably have at least one kind of functional group selected from a hydroxyl group, a glycidyl group and an amino group.
In a still another aspect of the present invention, there is provided a packing for chromatography, which is comprised of the above-mentioned cation exchanger.
In a further aspect of the present invention, there is provided a column for chromatography, which is packed with the above-mentioned packing.